Expression of recombinant proteinase K from Tritirachium album in yeast

ABSTRACT

The invention concerns a method for the expression of a gene coding for a soluble proteinase K in yeast e.g. in  Pichia pastoris  with subsequent secretion into the culture medium. In addition a method for purifying the heterologously expressed and secreted proteinase K is described.

The present invention concerns a method for the production ofrecombinant proteinase K in a soluble and active form in economicallyrelevant amounts.

Proteinase K (E.C. 3.4.21.64, also known as endopeptidase K) is anextracellular endopeptidase which is synthesized by the fungusTritirachium album Limber. It is a member of the class of serineproteases with the typical catalytic triad Asp³⁹-His⁶⁹-Ser²²⁴ (Jany, K.D. et al. (1986) FEBS Letters Vol. 199(2), 139-144). Since the sequenceof the polypeptide chain of 279 amino acids in length (Gunkel, F. A. andGassen, H. G. (1989) Eur. J. Biochem. Vol. 179(1), 185-194) and thethree dimensional structure (Betzel, C. et al. (1988) Eur. J. Biochem.Vol. 178(1), 155-71) has a high degree of homology to bacterialsubtilisins, proteinase K is classified as a member of the subtilisinfamily (Pahler, A. et al. (1984) EMBO J. Vol. 3(6), 1311-1314; Jany, K.D. and Mayer, B. (1985), Biol. Chem. Hoppe-Seyler, Vol. 366(5),485-492). Proteinase K was named on the basis of its ability tohydrolyse native keratin and thus allows the fungus to grow on keratinas the only source of carbon and nitrogen (Ebeling, W. et al. (1974)Eur. J. Biochem. Vol. 47(1), 91-97). Proteinase K has a specificactivity of more than 30 U/mg and is thus one of the most active of theknown endopeptidases (Betzel, C. et al. (1986) FEBS Lett. Vol. 197(1-2),105-110) and unspecifically hydrolyses native and denatured proteins.

Proteinase K from Tritirachium album Limber is translated in its naturalhost as a preproprotein. The sequence of the cDNA of the gene whichcodes for proteinase K was decoded in 1989 by Gunkel, F. A. and Gassen,H. G. (1989) Eur. J. Biochem. Vol. 179(1), 185-194. According to thisthe gene for prepro-proteinase K is composed of two exons and codes fora signal sequence of 15 amino acids in length, a prosequence of 90 aminoacids in length and a mature proteinase K of 279 amino acids in length.A 63 bp intron is located in the region of the prosequence. Theprepeptide is cleaved off during translocation into the endoplasmaticreticulum (ER). At present very little is known about the subsequentprocessing to form mature proteinase K with cleavage of the propeptide.

Consequently mature proteinase K consists of 279 amino acids. Thecompact structure is stabilized by two disulfide bridges and two boundcalcium ions. This explains why proteinase K compared to othersubtilisins has a considerably higher stability towards extreme pHvalues, high temperatures, chaotropic substances and detergents(Dolashka, P. et al. (1992) Int. J. Pept. Protein. Res. Vol. 40(5),465-471). Proteinase K is characterized by a high thermostability (up to65° C., Bajorath et al. (1988), Eur. J. Biochem. Vol. 176, 441-447) anda wide pH range (pH 7.5-12.0, Ebeling, W. et al. (1974) Eur. J. Biochem.Vol. 47(1), 91-97). Its activity is increased in the presence ofdenaturing substances such as urea or SDS (Hilz, H. et al. (1975) J.Biochem. Vol. 56(1), 103-108; Jany, K. D. and Mayer, B. (1985) Biol.Chem. Hoppe-Seyler, Vol. 366(5), 485-492).

The above-mentioned properties make proteinase K of particular interestfor biotechnological applications in which an unspecific proteindegradation is required. Special examples are nucleic acid isolation(DNA or RNA) from crude extracts and sample preparation in DNA analysis(Goldenberger, D. et al. (1995) PCR Methods Appl. Vol. 4(6), 368-370;U.S. Pat. No. 5,187,083; U.S. Pat. No. 5,346,999). Other applicationsare in the field of protein analysis such as structure elucidation.

Proteinase K is obtained commercially in large amounts by fermentationof the fungus Tritirachium album Limber (e.g. CBS 348.55, Merck strainNo. 2429 or the strain ATCC 22563). However, in this process theproduction of proteinase K is suppressed by glucose or free amino acids.Since protein-containing media also induce the expression of proteases,it is necessary to use proteins such as BSA, milk powder or soybeanflour as the only nitrogen source. The secretion of the protease startsas soon as the stationary phase of growth is reached (Ebeling, W. et al.(1974) Eur. J. Biochem. Vol. 47(1), 91-97).

Since Tritirachium album Limber is consequently unsuitable forfermentation on a large scale and moreover is difficult to geneticallymanipulate, various attempts have been made to overexpress recombinantproteinase K in other host cells. However, no significant activity wasdetected in these experiments due to lack of expression, formation ofinactive inclusion bodies or problems with the renaturation (Gunkel, F.A. and Gassen, H. G. (1989) Eur. J. Biochem. Vol. 179(1), 185-194;Samal, B. B. et al. (1996) Adv. Exp. Med. Biol. Vol. 379, 95-104).

Tritirachium album Limber is a slowly growing fungus which only secretessmall amounts of proteases into the medium. It has the disadvantage of aslower cell cycle compared to yeast and the lower optical density thatcan be achieved in a fermenter. In addition it is known that T. albumalso produces other proteases apart from proteinase K which cancontaminate the preparation (Samal, B. B. et al. (1991) Enzyme Microb.Technol. Vol. 13, 66-70).

Although in principle it is possible to express proteinase K in E. coli,it is not expressed in a soluble form but in so-called inclusion bodiesfrom which the enzyme has to be subsequently solubilized and renaturedby certain measures. A disadvantage of this method is that a lot ofprotein is lost during the solubilization and renaturing.

Hence the object of the present invention is to provide a method forproducing recombinant proteinase K in economically relevant amounts.

It has surprisingly turned out that it is possible to express andsecrete recombinant proteinase K as a zymogenic precursor in a solubleform in yeast which is autocatalytically activated to form activeproteinase K. Another subject matter of the invention is thepurification of active proteinase K from the medium supernatant.

Hence the present invention concerns a method for producing recombinantproteinase K comprising the steps:

-   a) transformation of a host cell with a vector containing a DNA    coding for the zymogenic precursor of proteinase K which is fused    upstream of the coding sequence with a sequence in the reading frame    which codes for a signal peptide and is under the control of a    suitable promoter for the host cell,-   b) expression of the zymogenic precursor of proteinase K-   c) secretion and autocatalytic activation of proteinase K-   d) isolation and purification of proteinase K, characterized in that    the host cell is a yeast cell and the protein is secreted in a    soluble form by this expression host.

In a special embodiment of the method according to the invention thehost cell is selected from the following group: Pichia species,Hansenula species such as Hansenula polymorpha, Saccharomyces species,Schizosaccharomyces species, Yarrowia species such as Yarrowialipolytica, Kluyveromyces species and Aspergillus species. It isparticularly preferred according to the invention when Pichia pastorisis used as the host cell.

Furthermore it has proven to be advantageous for the method according tothe invention when the host cell is transformed with a DNA coding forthe zymogenic precursor and the proteinase K is autocatalyticallyactivated at a later time during or immediately after secretion into theculture medium.

When using Pichia pastoris as a host cell, the gene coding for thezymogenic precursor of proteinase K is preferably cloned into thefollowing vectors: pPICZ, pPICZα, pGAPZ, pGAPZα, pPICZαA and pPIC9K. Inthis case the vectors: pPICZαA and pPIC9K are particularly preferred.According to the invention the vector pPICZαA is particularly preferred.The above-mentioned vectors are commercially available (Invitrogen).

In addition in the inventive method for producing recombinant proteinaseK it is preferred that the expression of proteinase K or the zymogenicprecursor of proteinase K is induced by methanol (pPIC vectors). Anothermethod is to induce the expression by glyceraldehyde phosphate (pGAPvectors).

In the inventive method for producing recombinant proteinase K thesecretion of the protein is preferably initiated by the N-terminalfusion of the signal peptide of the α-factor from Saccharomycescerevisiae. This for example means that the above-mentioned α-labelledvectors have the nucleotide sequence for the signal peptide of theα-factor from Saccharomyces cerevisiae. A fusion protein consisting ofthe signal peptide at the N-terminus and the target protein is thenproduced during translation. Another possible signal peptide would bethe natural signal sequence for proteinase K.

Furthermore it has proven to be particularly advantageous for theproduction of recombinant proteinase K, to transform the host cellPichia pastoris with the expression vectors pPICZαA and pPIC9K whichcontain a DNA coding for the zymogenic precursor and that the gene isunder the control of the AOX1 promoter and optionally of the AOX1terminator.

The present invention also concerns a vector containing a DNA coding forthe zymogenic precursor of proteinase K which is fused upstream of thecoding sequence with a sequence in the reading frame which codes for asuitable signal peptide and wherein the coding gene is under the controlof a suitable promoter and optionally terminator for the host cell andwherein this vector is suitable for the transformation of this hostcell. According to the invention the host cell is a yeast.

Hence the invention also concerns a recombinant vector which containsone or more copies of the recombinant DNA defined above. The vector ispreferably a plasmid which has a strong promoter for the host cell and asuitable signal peptide for the host cell for secreting proteins.Moreover it is also possible to fuse the native signal peptide ofprepro-proteinase K to the N-terminus of the propeptide as shown in SEQID NO.: 21 (signal sequence 1-15 (15 amino acids); prosequence 16-104(90 amino acids); sequence of the mature proteinase K 106-384 (279 aminoacids)). Methods are used to produce the expression vector which arefamiliar to a person skilled in the art and are described for example inSambrook et al. (1989).

Another subject matter of the present invention is a host celltransformed with one of the vectors listed above where the host cell isa yeast. The host cell is preferably selected from the following group:Pichia species, Hansenula species such as Hansenula polymorpha,Saccharomyces species, Schizosaccharomyces species, Yarrowia speciessuch as Yarrowia lipolytica, Kluyveromyces species and Aspergillusspecies. Pichia pastoris is particularly preferred as the host cell. Inparticular it is preferred when several vectors (each with one copy ofthe ppK gene) are integrated into the genome.

In addition the present invention concerns a method for purifyingproteinase K. In order to purify the protease the yeast cells areremoved in a first step by microfiltration or centrifugation. Theresulting clear solution contains the protease. This is followed by arebuffering by means of ultrafiltration in order to bind the product toa cation exchanger such as SP-Sepharose or SP-Sephadex (Pharmacia) orSP-Toyopearl (Tosoh Corporation). After the elution it is againrebuffered by means of ultrafiltration and bound to an anion exchangersuch as DEAE-Sepharose or Q-Sepharose (Pharmacia) or DEAE-Fraktogel(Merck). After another elution the pure protease is transferred by meansof ultrafiltration into a stable buffer system (Protein Purification,Principles and Practice, Robert K. Scopes, Springer Verlag, 1982).However, a person skilled in the art can use other methods ofpurification which are part of the prior art.

The method according to the invention surprisingly enables thepreparation of recombinant proteinase K in which the enzyme is producedby a heterologous host cell in a soluble and active form. The expressionof proteinase K with subsequent secretion of the enzyme into the culturemedium is of particular advantage since it prevents proteinase K fromdeveloping a strongly toxic effect in the cytosol of the host cell.Furthermore this ensures the correct formation of the two disulfidebridges which could not readily occur in the reducing environment of thecytosol. Hence an important advantage of the method according to theinvention is that it provides an approach for the soluble and activeproduction of a recombinant proteinase K. It is very surprising andinexplicable that the surface proteins of the host cells according tothe invention are not hydrolysed by a secreted proteinase K. Such anexpected hydrolysis of the surface proteins by proteinase K wouldinterfere with the life cycle of the host cell.

A proteinase K is obtained by the method according to the inventionwhich is homogeneous and hence particularly suitable for analytical anddiagnostic applications. The zymogenic precursor of proteinase Kaccording to the invention can optionally contain additional N-terminalmodifications and in particular sequences which facilitate purificationof the target protein (affinity tags). In addition the zymogenicprecursor can contain sequences which increase the efficiency oftranslation, which increase the folding efficiency and/or also sequenceswhich result in a secretion of the target protein into the culturemedium (natural presequence and other signal peptides).

Proteinase K in the sense of the invention means the sequence accordingto Gassen et al. (1989) shown in SEQ ID NO:1 as well as other variantsof proteinase K from Tritirachium album Limber like the amino acidsequence disclosed by Ch. Betzel et al. (Biochemistry 40 (2001),3080-3088) and in particular proteinase T (Samal, B. B. et al. (1989)Gene Vol. 85(2), 329-333; Samal, B. B. et al. (1996) Adv. Exp. Med.Biol. Vol. 379, 95-104) and proteinase R (Samal, B. B. et al. (1990)Mol. Microbiol. Vol. 4(10), 1789-1792; U.S. Pat. No. 5,278,062) and inaddition variants produced by recombinant means (as described forexample in WO 96/28556). SEQ ID NO:1 comprises a prosequence (1-90; 90amino acids) and the sequence of the mature proteinase K (91-368; 279amino acids). The proteinase K amino acid sequence described by Betzelet al. (Biochemistry 40 (2001), 3080-3088) has in particular aspartateinstead of a serine residue at position 207 of the active protease.

Pro-proteinase K in the sense of the invention means in particular aproteinase K whose N-terminus is linked to its prosequence according toSEQ ID NO: 1. In the case of subtilisin E which is closely related toproteinase K and variants thereof, the prosequence has an importantinfluence on the folding and formation of active protease (Ikemura, H.et al. (1987) J. Biol. Chem. Vol. 262(16), 7859-7864). In particular itis postulated that the prosequence acts as an intramolecular chaperone(Inouye, M. (1991) Enzyme Vol. 45, 314-321). After the folding it isprocessed to form the mature subtilisin protease by autocatalyticallycleaving the propeptide (Ikemura, H. and Inouye, M. (1988) J. Biol.Chem. Vol. 263(26), 12959-12963). This process occurs in the case ofsubtilisin E (Samal, B. B. et al. (1989) Gene Vol. 85(2), 329-333;Volkov, A. and Jordan, F. (1996) J. Mol. Biol. Vol. 262, 595-599),subtilisin BPN′ (Eder, J. et al. (1993) Biochemistry Vol. 32, 18-26),papain (Vernet, T. et al. (1991) J. Biol. Chem. Vol. 266(32),21451-21457) and thermolysin (Marie-Claire, C. (1998) J. Biol. Chem.Vol. 273(10), 5697-5701).

Only certain core regions of the prosequence which are usuallyhydrophobic appear to be necessary for the chaperone function since awide range of mutations have no influence on the activity (Kobayashi, T.and Inouye, M. (1992) J. Mol. Biol. Vol. 226, 931-933). In addition itis known that propeptides can be interchanged between various subtilisinvariants. Thus for example subtilisin BPN′ also recognizes theprosequence of subtilisin E (Hu, Z. et al. (1996) J. Biol. Chem. Vol.271(7), 3375-3384).

Hence the present invention concerns the prosequence according to SEQ IDNO:1 of 90 amino acids in length as well as other variants whichfacilitate folding. It also concerns a propeptide which is addedexogenously for the folding of mature proteinase K and has the functionsdescribed above.

Hence an important advantage of the method according to the invention isthat the recombinant proteinase K is secreted by an expression host intothe culture medium in a soluble and active form. Moreover the expressionhost used in the method according to the invention is not damaged orotherwise impaired by the very active and unspecific protease i.e. inparticular it continues to grow without problems and an increased celllysis is not observed. Furthermore the expression host according to theinvention is easier to handle compared to Tritirachium album and ischaracterized by higher growth rates.

DESCRIPTION OF THE FIGURES

FIG. 1

Expression plasmid pPICPK-1. A sequence coding for the zymogenic proformof proteinase K cloned into the starting vector pPICZαA (Invitrogen).

FIG. 2

Expression plasmid pPICPK-2. A sequence coding for the zymogenic proformof proteinase K cloned into the starting vector pPIC9K (Invitrogen).

EXAMPLES Example 1

Gene Synthesis

The gene for mature proteinase K from Tritirachium album Limber withouta signal sequence and without an intron was generated by means of genesynthesis. The sequence (without a native signal peptide) of Gunkel andGassen, 1989 of 368 amino acids in length was used as a template. Acodon-optimized nucleic acid sequence for expression in E. coli as wellas yeast was obtained by retranslating the amino acid sequence. Theamino acid sequence is shown in SEQ ID NO:1 and the nucleotide sequenceis shown in SEQ ID NO:2.

For the gene synthesis the gene was divided into 18 fragments of senseand reverse, complementary counterstrand oligonucleotides in alternatingsequence (SEQ ID NO: 3-20). An at least 15 bp region was attached to the5′ end and to the 3′ end which in each case overlapped the neighbouringoligonucleotides. Recognition sites for restriction endonucleases wereattached to the 5′ and 3′ ends of the synthetic gene outside the codingregion for subsequent cloning into expression vectors. Theoligonucleotide shown in SEQ ID NO: 3 which contains an EcoRI cleavagesite was used as a 5′ primer for cloning the pro-proteinase K gene. SEQID NO: 20 shows the 3′ primer containing a HindIII cleavage site. The 3′primer contains an additional stop codon after the natural stop codon toensure termination of the translation.

The oligonucleotides were linked together by means of a PCR reaction andthe resulting gene was amplified. For this the gene was firstly dividedinto three fragments of 6 oligonucleotides each and the three fragmentswere linked together in a second PCR cycle.

Fragment 1 is composed of the oligonucleotides shown in SEQ ID NO: 3-8,fragment 2 is composed of the oligonucleotides shown in SEQ ID NO: 9-14and fragment 3 is composed of the oligonucleotides shown in SEQ ID NO:15-20.

The following PCR parameters were applied PCR Reaction 1 (Generation ofThree Fragments)

5 min 95° C. hot start 2 min 95° C. 2 min 42° C. 30 cycles 1.5 min   72°C. {close oversize brace} 7 min 72° C. final extension

PCR Reaction 2 (Linkage of the Fragments to Form the Total Gene)

5 min 95° C. hot start 1.5 min   95° C. 2 min 48° C. {close oversizebrace} 6 cycles (without terminal primers) 2 min 72° C.addition of terminal primers

1.5 min 95° C. 1.5 min 60° C. {close oversize brace} 25 cycles (withterminal primers)   2 min 72° C.   7 min 72° C. final extension

Example 2

Cloning of the Synthetic Proteinase K Fragment from the Gene Synthesis

The PCR mixture was applied to an agarose gel and the ca. 1130 bp PCRfragment was isolated from the agarose gel (Geneclean II Kit from Bio101, Inc. CA USA). The fragment was cleaved for 1 hour at 37° C. withthe EcoRI and HindIII restriction endonucleases (Roche Diagnostics GmbH,Germany). At the same time the pUC18 plasmid (Roche Diagnostics GmbH,Germany) was cleaved for 1 hour at 37° C. with the EcoRI and HindIIIrestriction endonucleases, the mixture was separated by agarose gelelectrophoresis and the 2635 bp vector fragment was isolated.Subsequently the PCR fragment and the vector fragment were ligatedtogether using T4 DNA ligase. For this 1 μl (20 ng) vector fragment and3 μl (100 ng) PCR fragment, 1 μl 10× ligase buffer (Maniatis et al.,1989, B.27), 1 μl T4 DNA ligase, 4 μl sterile redistilled H₂O werepipetted, carefully mixed and incubated overnight at 16° C.

The cloned gene was examined by restriction analysis and by multiplesequencing of both strands.

Example 3

Vector Construction

The synthetic gene has to be firstly isolated again from the pUCplasmid. For this purpose 1 μg plasmid DNA was firstly incubated withthe restriction endonuclease HindIII (Roche Diagnostics GmbH) accordingto the manufacturer's instructions and subsequently the restrictionendonuclease was inactivated by heating to 65° C. for 20 min. Afterwardsthe resulting DNA overhangs were filled in with Klenow polymeraseaccording to the manufacturer's instructions (Roche Diagnostics GmbH)and the Klenow polymerase was then inactivated by incubating at 75° C.for 10 min. Finally the vector fragment which was now linearized of theabove-mentioned pUC plasmid was cleaved with the restrictionendonuclease EcoRI (Roche Diagnostics GmbH) according to themanufacturer's instructions, the reaction mixture was applied to a 1%agarose gel and the fragments were separated according to size byapplying a current (100 V/150 mA). The ca. 1120 bp fragment containingthe gene for pro-proteinase K (ppk gene) was isolated from the agarosegel (QIAquick Gel Extraction Kit/Qiagen).

The vector pPICZαA (Invitrogen) was cleaved with the restrictionendonuclease Asp718I (Roche Diagnostics GmbH) according to themanufacturer's instructions and the restriction endonuclease wasinactivated by heating the incubation mixture to 65° C. for 20 min.Afterwards the resulting DNA overhangs were filled in with Klenowpolymerase according to the manufacturer's instructions (RocheDiagnostics GmbH) and the Klenow polymerase was then inactivated byincubating at 75° C. for 10 min. Finally the vector fragment which wasnow linearized of pPICZαA was cleaved with the restriction endonucleaseEcoRI (Roche Diagnostics GmbH) according to the manufacturer'sinstructions, the reaction mixture was applied to a 1% agarose gel andthe fragments were separated according to size by applying a current(100 V/150 mA). The ca. 3550 bp vector fragment was isolated from theagarose gel (QIAquick Gel Extraction Kit/Qiagen).

The fragments obtained in this manner were ligated together by standardmethods (Sambrook et al. 1989). In this vector the ppk gene is under thecontrol of the AOX-1 promoter (promoter for alcohol oxidase 1 fromPichia pastoris, inducible with methanol) and is cloned using thiscloning strategy in the correct reading frame behind the signal peptideof the α-factor from Saccharomyces cerevisiae. The gene fragmentinserted in this manner was then examined for an error free sequence bymeans of restriction analysis and sequencing. The resulting expressionvector which contains the ppk gene which codes for pro-proteinase K wasnamed pPICPK-1 (see FIG. 1).

Subsequently the ppk gene was also cloned into pPIC9K (Invitrogen). Forthis purpose the vector pPICPK-1 was cleaved according to themanufacturer's instructions with the restriction endonucleases PmeI andNotI (Roche Diagnostics GmbH), the fragments from the restrictionmixture were separated according to size in a 1% agarose gel and the ca.1960 bp fragment containing the 3′ part of the AOX1-promoter region, thesequence for the signal peptide of the α-factor and the ppk gene wasisolated from the gel (QIAquick Gel Extraction Kit/Qiagen). At the sametime the vector pPIC9K was cleaved with the restriction endonucleasesPmeI and NotI (Roche Diagnostics GmbH) according to the manufacturer'sinstructions, the fragments from the restriction mixture were separatedaccording to size in a 1% agarose gel and the ca. 8450 bp vectorfragment was isolated from the gel (QIAquick Gel Extraction Kit/Qiagen).

Subsequently the fragments obtained in this manner were ligated togetherby standard methods (Sambrook et al. 1989). In this vector the ppk geneis also under the control of the AOX1-promoter (promoter for the alcoholoxidase 1 from Pichia pastoris, inducible with methanol). The vectorpPIC9K differs from the vector pPICZαA by the selection marker and bythree possibilities known to a person skilled in the art for integratingit into the Pichia genome depending on the vector linearization beforetransformation whereas the integration of pPICZαA into the AOX1-locus isfixed. The inserted gene fragment was then examined for an error-freesequence by means of restriction analysis and sequencing.

The resulting expression vector which contains the ppk gene which codesfor pro-proteinase K was named pPICPK-2 (see FIG. 2).

Example 4

Transformation of pPICPK-1 in Pichia pastoris

In order to transform pPICPK-1 in Pichia pastoris X-33 with subsequentintegration into the genome, the vector was firstly linearized with PmeI(Roche Diagnostics GmbH). The transformation was carried out by means ofelectroporation using a Gene Pulser II (Biorad).

For this purpose 5 ml YPD medium (according to the Invitrogen catalogue)was inoculated with a colony of Pichia pastoris wild-type strain andincubated overnight at 30° C. while shaking. The overnight culture wassubsequently reinoculated 1:2000 in 200 ml fresh YPD medium (accordingto the Invitrogen catalogue) and incubated overnight at 30° C. whileshaking until the OD₆₀₀ reached 1.3-1.5. The cells were centrifuged(1500×g/5 minutes) and the pellet was resuspended in 200 ml ice-coldsterile water (0° C.). The cells were again centrifuged (1500×g/5minutes) and resuspended in 100 ml ice-cold sterile water (0° C.). Thecells were again centrifuged and resuspended in 10 ml ice-cold (0° C.) 1M sorbitol (ICN). The cells were again centrifuged and resuspended in0.5 ml ice-cold (0° C.) 1 M sorbitol (ICN). The cells obtained in thismanner were kept on ice and used immediately for transformation.

About 1 μg linearized pPICPK-1 vector DNA was added to 80 μl of thecells and the entire mixture was transferred to an ice-cold (0° C.)electroporation cuvette and incubated for a further 5 minutes on ice.Subsequently the cuvette was transferred to a Gene Pulser II (Biorad)and the transformation was carried out at 1 kV, 1 kΩ and 25 μF. Afterelectroporation 1 ml 1 M sorbitol (ICN) was added to the mixture andsubsequently 100-150 μl was plated out on a YPDS agar plate (accordingto the Invitrogen catalogue) containing 100 μg/ml Zeocin® (Invitrogen).The plates were subsequently incubated for 2-4 days at 30° C.

Minimal dextrose grid plates were inoculated with the clones and theclones were analysed further.

Clones that had grown were picked, resuspended in 20 μl sterile waterand lysed with 17.5 U lyticase (Roche Diagnostics GmbH) (1 hour, 37° C.)and examined directly by means of PCR for the correct integration of theppk expression cassette.

Clones which had integrated the complete expression cassette duringtransformation into the genome were then used in expression experiments.

Example 5

Transformation of pPICPK-2 in Pichia pastoris

In order to transform pICPK-2 in Pichia pastoris GS115 with subsequentintegration into the genome, the vector was firstly linearized forvariant I with PmeI (Roche Diagnostics GmbH) to integrate it into theAOXI-locus and linearized with SalI (Roche Diagnostics GmbH) for variantII to integrate it into the His4 locus. The transformation was carriedout by means of electroporation using a Gene Pulser (Biorad).

For this purpose 5 ml YPD medium (according to the Invitrogen catalogue)was inoculated with a colony of Pichia pastoris GS115 wild-type strainand incubated overnight at 30° C. while shaking. The overnight culturewas subsequently reinoculated 1:2000 in 200 ml fresh YPD medium(according to the Invitrogen catalogue) and incubated overnight at 30°C. while shaking until the OD₆₀₀ reached 1.3-1.5. The cells werecentrifuged (1500×g/5 minutes) and the pellet was resuspended in 200 mlice-cold sterile water (0° C.). The cells were again centrifuged(1500×g/5 minutes) and resuspended in 100 ml ice-cold sterile water (0°C.). The cells were again centrifuged and resuspended in 10 ml ice-cold(0° C.) 1 M sorbitol (ICN). The cells were again centrifuged andresuspended in 0.5 ml ice-cold (0° C.) 1 M sorbitol (ICN). The cellsobtained in this manner were kept on ice and used immediately fortransformation.

About 1 μg linearized pPICPK-2 vector DNA was added to 80 μl of thecells and the entire mixture was transferred to an ice-cold (0° C.)electroporation cuvette and incubated for a further 5 minutes on ice.Subsequently the cuvette was transferred to a Gene Pulser II (Biorad)and the transformation was carried out at 1 kV, 1 kΩ and 25 μF. Afterelectroporation 1 ml 1 M sorbitol (ICN) was added to the mixture andsubsequently 100-150 μl was plated out on a MM agar plate (minimalmedium according to the Invitrogen catalogue) without histidine. Theplates were subsequently incubated for 2-4 days at 30° C. Clones ofPichia pastoris GS115 which have a defective His4 gene caused bymutation (histidinol dehydrogenase) can only grow on these plates whenthey have integrated the vector pPICPK-2 which has a functional His4gene as an insert and can hence compensate the deficiency in histidinebiosynthesis.

Minimal dextrose grid plates were inoculated with the clones and theclones were analysed further. Clones that had grown were picked,resuspended in 20 μl sterile water and lysed with 17.5 U lyticase (RocheDiagnostics GmbH) (1 hour, 37° C.) and examined directly by means of PCRfor the correct integration of the ppk expression cassette.

Clones which had integrated the complete expression cassette duringtransformation into the genome were then used in expression experiments.

Example 6

Expression of Proteinase K

10 ml BMGY medium (according to the Invitrogen catalogue) was inoculatedwith positive clones and incubated overnight at 30° C. while shaking.Subsequently the optical density at 600 nm was determined and 10 ml BMMYmedium (according to the Invitrogen catalogue) was inoculated in such amanner that an OD₆₀₀ of 1 resulted. The BMMY medium (according to theInvitrogen catalogue) contains methanol (Mallinckrodt Baker B.V) whichinduces the expression of proteinase K via the AOX1 promoter.

The shaking flask was incubated at 30° C. while shaking, samples weretaken every 24 hours, the OD₆₀₀ was determined, an activity test wascarried out for expression of proteinase K and each time 0.5% methanol(Mallinckrodt Baker B.V) was refed for further induction. The expressionexperiments ran for 168 hours.

Example 7

Activity Test for Secreted Recombinant Proteinase K

For the activity test for recombinant proteinase K one requiresCaCl₂×2H₂O (Merck ID-No. 102382), DMSO (Merck, ID-No. 109678), thesubstrate Suc-Ala-Ala-Pro-Phe-pNA (Roche Diagnostics ID-No. 0716766) andTris base (Roche Diagnostics ID-No. 0153265).

The composition of the solutions was as follows:

Solution 1: 50 mmol/l Tris-Base; 10 mmol/l CaCl₂ pH 8.2

Solution 2: 125 mg Suc-Ala-Ala-Pro-Phe-pNA dissolved in 1 ml DMSO

The cells were centrifuged (5 min 10000 rpm Eppendorf bench centrifuge)and the supernatant was diluted 1:500 in solution 1.

2 ml of solution 1 was pipetted into a cuvette and 0.02 ml of solution 2was added. Both solutions were mixed and incubated at a reactiontemperature of 25° C. The reaction was started by adding 0.05 ml of thediluted supernatant as stated above and remixing, the change inabsorbance at 405 nm was measured and the ΔA/min in the linear regionwas measured. The following formula was then used for the calculation:

${activity} = {\frac{2.07}{\varepsilon \times 1 \times 0.05}\Delta\; A\text{/}{\min\lbrack {U\text{/}{ml}\mspace{14mu}{sample}\mspace{14mu}{solution}} \rbrack}}$

2.07=sample volume

ε₄₀₅=10.4 [mmol⁻¹×1×cm⁻¹]

1=path length of the cuvette

0.05=volume of the added sample

1. A method for producing recombinant proteinase K comprising: (a)transforming a Pichia pastoris yeast cell with a vector constructcomprising a DNA sequence encoding a signal peptide fused upstream of aDNA sequence encoding a zymogenic precursor of proteinase K, andadditional sequences that place the zymogenic precursor coding sequenceunder the control of a promoter for the host cell, (b) expressing thezymogenic precursor of proteinase K, and (c) secreting andautocatalytically activating the zymogenic precursor to produce saidrecombinant proteinase K, wherein the proteinase K is secreted insoluble form, and the yeast cells continue to grow during recombinantproteinase K expression and secretion, without a detectable increase inyeast cell lysis.
 2. The method of claim 1 wherein the DNA sequenceencoding the zymogenic precursor of proteinase K is cloned into a vectorselected from the group consisting of pPICZ, pPICZα, pGAPZ, pGAPZα,pPICZαA, and pPIC9K.
 3. The method of claim 1 wherein the expressing ofthe zymogenic precursor of proteinase K is induced by methanol.
 4. Themethod of claim 1 wherein the vector construct comprises a nucleic acidsequence encoding the signal peptide of α-factor from Saccharomycescerevisiae.
 5. The method of claim 1 wherein the vector is pPICZαA andthe promoter is AOXI.
 6. A method for producing recombinant proteinase Kcomprising expressing a zymogenic precursor of proteinase K in a Pichiapastoris yeast cell, said yeast cell comprising a vector constructcomprising a DNA sequence encoding a signal peptide fused upstream of aDNA sequence encoding a zymogenic precursor of proteinase K, andadditional sequences that place the zymogenic precursor coding sequenceunder the control of a promoter for the yeast cell, secreting andautocatalytically activating the zymogenic precursor to produce saidrecombinant proteinase K, wherein the proteinase K is secreted insoluble form, and the yeast cells continue to grow during recombinantproteinase K expression and secretion, without a detectable increase inyeast cell lysis.
 7. The method of claim 1 wherein the vector isselected from the group consisting of pPICZα and pPIC9K.
 8. The methodof claim 1 wherein the DNA sequence coding for a zymogenic precursor ofproteinase K comprises the sequence of SEQ ID NO: 2.